Serum Transferrin is the protein responsible for the transport of Fe3+ between sites of uptake, utilization, and storage. It also binds Ca3+, In3+, and Al3+ when these metal ions enter the blood. Characterization of the interactions of these metal ions with transferrin is important for understanding the mechanism of intracellular iron release, for the development of new drugs for treating iron overload, and for understanding the pharmacology and toxicology of compounds of the group 13 metal ions. Based on previous results from this project, it is proposed that iron removal proceeds through two parallel pathways, one which appears to be limited by a conformational change in the protein, and a second pathway which is first-order in ligand. The proposed research will further evaluate this mechanism versus alternative proposals from other labs. Rates of metal ion removal for Fe3+, Al3+, Ga3+, and In3+ will be followed using electronic spectroscopy and electrophoresis to determine the effect of the metal ion on absolute rates of exchange and on the relative importance of the two pathways. Detailed comparisons between neutral and anionic ligands will be made to assess the role of anionic ligands as allosteric modifiers of transferrin. The effects of inorganic salts on the two pathways for iron removal will be measured, and anion-induced conformational changes in transferrin will be characterized by circularly polarized luminescence of lanthanide-Tf complexes. Additional studies of anion and metal binding to apotransferrin at the low pH associated with intracellular iron release will be conducted to determine whether anions can promote iron release by thermodynamic competition for a common binding site. It is proposed that cationic amino acid side chains near the metal-binding site may be the locus for selective anion effects on the kinetics of iron removal. Recombinant transferrins will be prepared in which residues suspected to be involved in anion binding will be selectively altered. The thermodynamics of anion binding to the recombinant proteins will be measured to determine the residues involved in anion binding, and the kinetics of iron release from the recombinant proteins to a series of neutral and anionic ligands will be evaluated. In addition, the effect of ligands and non-coordinating anions on the rate of exchange of the synergistic bicarbonate anion in Fe-HCO3-Tf will be followed by a new technique involving mass spectral detection of [13C) CO2.